Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

CDCA7 promotes TGF-ß-induced epithelial-mesenchymal transition via transcriptionally regulating Smad4/Smad7 in ESCC.

Cell division cycle associated 7 (CDCA7) is a copy number amplification gene that contributes to the metastasis and invasion of tumors, including esophageal squamous cell carcinoma (ESCC). This present study aimed at clarifying whether high expression of CDCA7 promotes the metastasis and invasion of ESCC cell lines and exploring the underlying mechanisms implicated in epithelial-mesenchymal transition (EMT) of ESCC. The role of CDCA7 in the regulation of ESCC metastasis and invasion was evaluated using ESCC cell lines. Expression of EMT-related markers including E-cadherin, N-cadherin, Vimentin, Snail, and Slug, transforming growth factor ß (TGF-ß) signaling pathway including Smad2/3, p-Smad2/3, Smad4, and Smad7 were detected in CDCA7 knockdown and overexpressed cell lines. Dual-luciferase reporter assay and rescue assay were used to explore the underlying mechanisms that CDCA7 contributed to the metastasis and invasion of ESCC. High CDCA7 expression significantly promoted the metastasis and invasion of ESCC cell lines both in vivo and in vitro. Additionally, the expression of CDCA7 positively correlated with the expression of N-cadherin, Vimentin, Snail, Slug, TGF-ß signaling pathway and negatively correlated with the expression of E-cadherin. Furthermore, CDCA7 transcriptionally regulated the expression of Smad4 and Smad7. Knockdown of CDCA7 inhibited the TGF-ß signaling pathway and therefore inhibited EMT. Our data indicated that CDCA7 was heavily involved in EMT by regulating the expression of Smad4 and Smad7 in TGF-ß signaling pathway. CDCA7 might be a new therapeutic target in the suppression of metastasis and invasion of ESCC.

A microRNA-based liquid biopsy signature for the early detection of esophageal squamous cell carcinoma: a retrospective, prospective and multicenter study.

BACKGROUND: Currently, there is no clinically relevant non-invasive biomarker for early detection of esophageal squamous cell carcinoma (ESCC). Herein, we established and evaluated a circulating microRNA (miRNA)-based signature for the early detection of ESCC using a systematic genome-wide miRNA expression profiling analysis. METHODS: We performed miRNA candidate discovery using three ESCC tissue miRNA datasets (n = 108, 238, and 216) and the candidate miRNAs were confirmed in tissue specimens (n = 64) by qRT-PCR. Using a serum training cohort (n = 408), we conducted multivariate logistic regression analysis to develop an ESCC circulating miRNA signature and the signature was subsequently validated in two independent retrospective and two prospective cohorts. RESULTS: We identified eighteen initial miRNA candidates from three miRNA expression datasets (n = 108, 238, and 216) and subsequently validated their expression in ESCC tissues. We thereafter confirmed the overexpression of 8 miRNAs (miR-103, miR-106b, miR-151, miR-17, miR-181a, miR-21, miR-25, and miR-93) in serum specimens. Using a serum training cohort, we developed a circulating miRNA signature (AUC:0.83 [95%CI:0.79-0.87]) and the diagnostic performance of the miRNA signature was confirmed in two independent validation cohorts (n = 126, AUC:0.80 [95%CI:0.69-0.91]; and n = 165, AUC:0.89 [95%CI:0.83-0.94]). Finally, we demonstrated the diagnostic performance of the 8-miRNA signature in two prospective cohorts (n = 185, AUC:0.92, [95%CI:0.87-0.96]); and (n = 188, AUC:0.93, [95%CI:0.88-0.97]). Importantly, the 8-miRNA signature was superior to current clinical serological markers in discriminating early stage ESCC patients from healthy controls (p < 0.001). CONCLUSIONS: We have developed a novel and robust circulating miRNA-based signature for early detection of ESCC, which was successfully validated in multiple retrospective and prospective multinational, multicenter cohorts.

An N-glycoproteomic site-mapping analysis reveals glycoprotein alterations in esophageal squamous cell carcinoma.

BACKGROUND: Aberrant glycosylation has been recognized as a hallmark of cancer and N-glycosylation is one of the main types of glycosylation in eukaryotes. Although N-glycoproteomics has made contributions to the discovery of biomarkers in a variety of cancers, less is known about the abnormal glycosylation signatures in esophageal squamous cell carcinoma (ESCC). METHODS: In this study, we reported the proteomics and N-glycoproteomic site-mapping analysis of eight pairs of ESCC tissues and adjacent normal tissues. With zic-HILIC enrichment, TMT-based isobaric labeling, LC-MS/MS analysis, differentially expressed N-glycosylation was quantitatively characterized. Lectin affinity enrichment combined with western blot was used to validate the potential biomarkers in ESCC. RESULTS: A series of differentially expressed glycoproteins (e.g., LAMP2, PLOD2) and enriched signaling pathways (e.g., metabolism-related pathway, ECM-receptor interaction, focal adhesion) were identified. Besides that, seven significantly enriched motifs were found from the identified N-glycosylation sites. Three clusters were identified after conducting the dynamic profiling analysis of glycoprotein change during lymph node metastasis progression. Further validation found that the elevated fucosylation level of ITGB1, CD276 contributed to the occurrence and development of ESCC, which might be the potential biomarkers in ESCC. CONCLUSION: In summary, we characterized the N-glycosylation and N-glycoprotein alterations associated with ESCC. The typical changes in glycoprotein expression and glycosylation occupancy identified in our study will not only be used as ESCC biomarkers but also improve the understanding of ESCC biology.

Promoter Methylation-Regulated miR-145-5p Inhibits Laryngeal Squamous Cell Carcinoma Progression by Targeting FSCN1.

Laryngeal squamous cell carcinoma (LSCC) is a common form of head and neck cancer with poor prognosis. However, the mechanism underlying the pathogenesis of LSCC remains unclear. Here, we demonstrated increased expression of fascin actin-bundling protein 1 (FSCN1) and decreased expression of microRNA-145-5p (miR-145-5p) in a clinical cohort of LSCC. Luciferase assay revealed that miR-145-5p is a negative regulator of FSCN1. Importantly, low miR-145-5p expression was correlated with TNM (tumor, node, metastasis) status and metastasis. Moreover, cases with low miR-145-5p/high FSCN1 expression showed poor prognosis, and these characteristics together served as independent prognostic indicators of survival. Gain- and loss-of-function studies showed that miR-145-5p overexpression or FSCN1 knockdown inhibited LSCC migration, invasion, and growth by suppressing the epithelial-mesenchymal transition along with inducing cell-cycle arrest and apoptosis. Additionally, hypermethylation of the miR-145-5p promoter suggested that repression of miR-145-5p arises through epigenetic inactivation. LSCC tumor growth in vivo could be inhibited by using miR-145-5p agomir or FSCN1 small interfering RNA (siRNA), which highlights the potential for clinical translation. Collectively, our findings indicate that miR-145-5p plays critical roles in inhibiting the progression of LSCC by suppressing FSCN1. Both miR-145-5p and FSCN1 are important potential prognostic markers and therapeutic targets for LSCC.

Cadmium inhibits apoptosis of human esophageal epithelial cells by upregulating CDK6.

Cadmium (Cd) exposure in environment is associated with development of esophageal cancer. However, the mechanisms of Cd-induced carcinogenesis are still not been fully cleared, and the present study aimed to explore the possible etiological mechanism of Cd-induced esophageal cancer. Human esophageal epithelial cell lines (HET-1A and KYSE450) were treated with CdCl2 at 0.05 mg/l for 12, 24 h, and the then the apoptosis were detected using flow cytometry with annexin-V-FITC/PI staining. Results showed that apoptosis of treatment groups was significantly inhibited, and decreased reactive oxygen species (ROS) production played a key role in the inhibitory effects by upregulating Bcl-2 and downregulating Caspase-3/9. The relief of oxidative stress during Cd exposure was actively promoted by the increased nicotinamide adenine dinucleotide phosphoric acid and glutathione levels. To investigate the causes of enhanced intracellular antioxidant capacity, the activity of pyruvate kinase (PK), a key enzyme of glycolysis, was detected. Our results showed that PK activity was inhibited, suggesting that glycolysis process was blocked which promoted more intermediate metabolites of glycolysis to be used for reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH) or other antioxidants synthesis. PK activity was closely correlated with phosphorylation of pyruvate kinase M2 (PKM2), and a highly negative correlation (correlation coefficients: -0.835, p < 0.05) between them was found. Western blotting showed the overphosphorylation of PKM2 in Cd-exposed cells, resulting from increased expression of cyclin-dependent kinases 6 (CDK6). These results suggested a possible mechanism of carcinogenic: Cd-induced upregulation of CDK6 in esophageal cell lines caused PKM2 overphosphorylation inhibiting PK activity, thereby shunting glucose-derived carbon into the pentose phosphate pathway and promoting the production of NADPH and reduced glutathione (GSH) to neutralize ROS, which finally results in the inhibited apoptosis.

FAT1, a direct transcriptional target of E2F1, suppresses cell proliferation, migration and invasion in esophageal squamous cell carcinoma.

OBJECTIVE: Growing evidence indicates that FAT atypical cadherin 1 (FAT1) has aberrant genetic alterations and exhibits potential tumor suppressive function in esophageal squamous cell carcinoma (ESCC). However, the role of FAT1 in ESCC tumorigenesis remains not well elucidated. The aim of this study was to further investigate genetic alterations and biological functions of FAT1, as well as to explore its transcriptional regulation and downstream targets in ESCC. METHODS: The mutations of FAT1 in ESCC were achieved by analyzing a combined study from seven published genomic data, while the copy number variants of FAT1 were obtained from an analysis of our previous data as well as of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases using the cBioPortal. The transcriptional regulation of FAT1 expression was investigated by chromatin immunoprecipitation (ChIP) and the luciferase reporter assays. In-cell western, Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the indicated gene expression. In addition, colony formation and Transwell migration/invasion assays were employed to test cell proliferation, migration and invasion. Finally, RNA sequencing was used to study the transcriptomes. RESULTS: FAT1 was frequently mutated in ESCC and was deleted in multiple cancers. Furthermore, the transcription factor E2F1 occupied the promoter region of FAT1, and depletion of E2F1 led to a decrease in transcription activity and mRNA levels of FAT1. Moreover, we found that knockdown of FAT1 promoted KYSE30 and KYSE150 cell proliferation, migration and invasion; while overexpression of FAT1 inhibited KYSE30 and KYSE410 cell proliferation, migration and invasion. In addition, knockdown of FAT1 led to enrichment of the mitogen-activated protein kinase (MAPK) signaling pathway and cell adhesion process. CONCLUSIONS: Our data provided evidence for the tumor suppressive function of FAT1 in ESCC cells and elucidated the transcriptional regulation of FAT1 by E2F1, which may facilitate the understanding of molecular mechanisms of the progression of ESCC.

Role of CXCR4 and SDF1 as prognostic factors for survival and the association with clinicopathology in colorectal cancer: A systematic meta-analysis.

C-X-C chemokine receptor type 4 and stromal cell-derived factor-1 were proven to play important roles in several types of cancer and in many biological processes connected with tumor growth, invasion, angiogenesis, and metastasis. However, the clinical significance of C-X-C chemokine receptor type 4 and stromal cell-derived factor-1 expression in colorectal cancer remains inaccurate. The purpose of this systematic meta-analysis is to investigate the role of C-X-C chemokine receptor type 4 and stromal cell-derived factor-1 as prognostic factors for survival and the association between C-X-C chemokine receptor type 4/ stromal cell-derived factor-1 and clinicopathology in colorectal cancer. Databases including PubMed, EMBASE, and Cochrane Library were searched for relevant literatures updated till January 2017. Review Manager 5.3 was used for data analysis. In our meta-analysis, C-X-C chemokine receptor type 4 expression is related to tumor-node-metastasis stage, tumor differentiation, liver metastasis, lymph node metastasis, distant metastasis, and diagnosis, and no correlation of C-X-C chemokine receptor type 4 expression with tumor size, gender, preoperative carcinoembryonic antigen, age, or vascular invasion has been observed. Stromal cell-derived factor-1 expression has no relationship with tumor-node-metastasis stage, lymph node metastasis, vascular invasion, age, gender, distant metastasis, or diagnosis. The expression of stromal cell-derived factor-1 has association with tumor differentiation. Moreover, the pooled hazard ratio for disease-free survival/overall survival showed that overexpression of C-X-C chemokine receptor type 4/stromal cell-derived factor-1 reduced disease-free survival/overall survival in colorectal cancer. Therefore, High expression of C-X-C chemokine receptor type 4/stromal cell-derived factor-1 which is essential in tumor progression can predict poor survival that may provide more advance prognostic clues to colorectal cancer patients.

Tag SNPs of CFI contributed to the susceptibility for non-small cell lung cancer in Chinese population.

Complement factor I (CFI) plays an important role in the development of non-small cell lung cancer (NSCLC). This study aims to examine the association of CFI genetic variants with the risk of developing NSCLC in Chinese population. A hospital-based case-control study was conducted in 470 patients with NSCLC and 470 controls in Chinese population. Totally, 13 tag single nucleotide polymorphisms (tag SNPs) of CFI were selected by Haploview software using the HapMap database. Genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY. The odds ratios (ORs) and 95% confidence interval (95% CI) were calculated by logistic regression model. Our results showed that individuals with rs6822976 GG genotype had a significant decreased risk of NSCLC (OR = 0.64; 95% CI = 0.42-0.98) when compared with rs6822976 AA genotype carriers. We also found that rs7671905 TT genotype exhibited a significant decreased risk of NSCLC compared with CC genotype with OR (95% CI) of 0.55 (0.33-0.91). There was no significant association between other selected SNPs and the risk of NSCLC. When stratified by smoking status, the decreased risk of NSCLC was observed to be associated with the genotype with at least one rs6822976 G allele among non-smokers (OR = 0.66; 95% CI = 0.47-0.93), but not among smokers (OR = 1.01; 95% CI = 0.67-1.53). For CFI rs7671905 polymorphism, the individuals with at least one T allele have a decreased risk of NSCLC with OR (95 % CI) of 0.71 (0.51-0.99), but not among smokers (OR = 0.93; 95% CI = 0.61-1.41). When stratified by age, we found that rs7671905 TT genotype has contributed to the decreased risk of NSCLC among older subjects with OR (95% CI) of 0.46 (0.23-0.95), but not among younger subjects with OR (95% CI) of 0.64 (0.31-1.34) (P(interaction) = 0.03). After stratifying by sex, our study showed that rs7671905 TT genotype was related to the risk of NSCLC among males (OR = 0.53; 95% CI = 0.29-0.98), but not among females (OR = 0.62; 95% CI = 0.25-1.57) (P(interaction) = 0.03). CFI genetic variants played an important role in the development of NSCLC in Chinese population.

IGH repertoire analysis at scale: deciphering the complexity of B cell infiltration and migration in esophageal squamous cell carcinoma.

Tumor-infiltrating B-lineage cells have become predictors of prognosis and immunotherapy responses in various cancers. However, limited knowledge about their infiltration and migration patterns has hindered the understanding of their anti-tumor functions. Here, we examined the immunoglobulin heavy chain (IGH) repertoires in 496 multi-regional tumor, 107 normal tissue, and 48 metastatic lymph node samples obtained from 107 patients with esophageal squamous cell carcinoma (ESCC). Our study revealed higher IgG-type B-lineage cells infiltration in tumors than in healthy tissue, which was associated with improved patient outcomes. Genes such as ACTN1, COL6A5, and pathways like focal adhesion, which shapes the physical structure of tumors, could affect B-lineage cell infiltration. Notably, the IGH sequence was used as an identity-tag to monitor B cell migration, and their infiltration schema within the tumor were depicted based on our multi-regional tumor specimens. This analysis revealed an escalation in B cell clones overlapped between metastatic lymph nodes and tumors. Therefore, the Lymph Node Activation Index was defined, which could predict the outcomes of patients with lymph node metastasis. This research introduces a novel framework for probing B cell infiltration and migration within the tumor microenvironment using large-scale transcriptome data, while simultaneously providing fresh perspectives on B cell immunology within ESCC.

Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.

BACKGROUND: Mitochondria play essential roles in tumorigenesis; however, little is known about the contribution of mitochondrial DNA (mtDNA) to esophageal squamous cell carcinoma (ESCC). Whole-genome sequencing (WGS) is by far the most efficient technology to fully characterize the molecular features of mtDNA; however, due to the high redundancy and heterogeneity of mtDNA in regular WGS data, methods for mtDNA analysis are far from satisfactory. METHODS: Here, we developed a likelihood-based method dMTLV to identify low-heteroplasmic mtDNA variants. In addition, we described fNUMT, which can simultaneously detect non-reference nuclear sequences of mitochondrial origin (non-ref NUMTs) and their derived artifacts. Using these new methods, we explored the contribution of mtDNA to ESCC utilizing the multi-omics data of 663 paired tumor-normal samples. RESULTS: dMTLV outperformed the existing methods in sensitivity without sacrificing specificity. The verification using Nanopore long-read sequencing data showed that fNUMT has superior specificity and more accurate breakpoint identification than the current methods. Leveraging the new method, we identified a significant association between the ESCC overall survival and the ratio of mtDNA copy number of paired tumor-normal samples, which could be potentially explained by the differential expression of genes enriched in pathways related to metabolism, DNA damage repair, and cell cycle checkpoint. Additionally, we observed that the expression of CBWD1 was downregulated by the non-ref NUMTs inserted into its intron region, which might provide precursor conditions for the tumor cells to adapt to a hypoxic environment. Moreover, we identified a strong positive relationship between the number of mtDNA truncating mutations and the contribution of signatures linked to tumorigenesis and treatment response. CONCLUSIONS: Our new frameworks promote the characterization of mtDNA features, which enables the elucidation of the landscapes and roles of mtDNA in ESCC essential for extending the current understanding of ESCC etiology. dMTLV and fNUMT are freely available from https://github.com/sunnyzxh/dMTLV and https://github.com/sunnyzxh/fNUMT , respectively.

Immune-tumor interaction dictates spatially directed evolution of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a poor-prognostic cancer type with extensive intra- and inter-patient heterogeneity in both genomic variations and tumor microenvironment (TME). However, the patterns and drivers of spatial genomic and microenvironmental heterogeneity of ESCC remain largely unknown. Here, we generated a spatial multi-omic atlas by whole-exome, transcriptome, and methylome sequencing of 507 tumor samples from 103 patients. We identified a novel tumor suppressor PREX2, accounting for 22% of ESCCs with frequent somatic mutations or hyper-methylation, which promoted migration and invasion of ESCC cells in vitro. Analysis of the TME and quantification of subclonal expansion indicated that ESCCs undergo spatially directed evolution, where subclones mostly originated from the tumor center but had a biased clonal expansion to the upper direction of the esophagus. Interestingly, we found upper regions of ESCCs often underwent stronger immunoediting with increased selective fitness, suggesting more stringent immune selection. In addition, distinct TMEs were associated with variable genomic and clinical outcomes. Among them, hot TME was associated with high immune evasion and subclonal heterogeneity. We also found that immunoediting, instead of CD8+ T cell abundance, acts as an independent prognostic factor of ESCCs. Importantly, we found significant heterogeneity in previously considered potential therapeutic targets, as well as BRCAness characteristics in a subset of patients, emphasizing the importance of focusing on heterogeneity in ESCC targeted therapy. Collectively, these findings provide novel insights into the mechanisms of the spatial evolution of ESCC and inform precision therapeutic strategies.

Genomic characterization and risk stratification of esophageal squamous dysplasia.

Objectives: The majority of esophageal squamous dysplasia (ESD) patients progress slowly, while a subset of patients can undergo recurrence rapidly or progress to invasive cancer even after proper treatment. However, the molecular mechanisms underlying these clinical observations are still largely unknown. Methods: By sequencing the genomic data of 160 clinical samples from 49 tumor-free ESD patients and 88 esophageal squamous cell carcinoma (ESCC) patients, we demonstrated lower somatic mutation and copy number alteration (CNA) burden in ESD compared with ESCC. Results: Cross-species screening and functional assays identified ACSM5 as a novel driver gene for ESD progression. Furthermore, we revealed that miR-4292 promoted ESD progression and could serve as a non-invasive diagnostic marker for ESD. Conclusions: These findings largely expanded our understanding of ESD genetics and tumorigenesis, which possessed promising significance for improving early diagnosis, reducing overtreatment, and identifying high-risk ESD patients.

Roles of Kruppel-associated Box (KRAB)-associated Co-repressor KAP1 Ser-473 Phosphorylation in DNA Damage Response.

The Kruppel-associated box (KRAB)-associated co-repressor KAP1 is an essential nuclear co-repressor for the KRAB zinc finger protein superfamily of transcriptional factors. Ataxia telangiectasia mutated (ATM)-Chk2 and ATM- and Rad3-related (ATR)-Chk1 are two primary kinase signaling cascades activated in response to DNA damage. A growing body of evidence suggests that ATM and ATR phosphorylate KAP1 at Ser-824 in response to DNA damage and regulate KAP1-dependent chromatin condensation, DNA repair, and gene expression. Here, we show that, depending on the type of DNA damage that occurs, KAP1 Ser-473 can be phosphorylated by ATM-Chk2 or ATR-Chk1 kinases. Phosphorylation of KAP1 at Ser-473 attenuated its binding to the heterochromatin protein 1 family proteins and inhibited its transcriptional repression of KRAB-zinc finger protein (KRAB-ZFP) target genes. Moreover, KAP1 Ser-473 phosphorylation induced by DNA damage stimulated KAP1-E2F1 binding. Overexpression of heterochromatin protein 1 significantly inhibited E2F1-KAP1 binding. Elimination of KAP1 Ser-473 phosphorylation increased E2F1-targeted proapoptotic gene expression and E2F1-induced apoptosis in response to DNA damage. Furthermore, loss of phosphorylation of KAP1 Ser-473 led to less BRCA1 focus formation and slower kinetics of loss of γH2AX foci after DNA damage. KAP1 Ser-473 phosphorylation was required for efficient DNA repair and cell survival in response to DNA damage. Our studies reveal novel functions of KAP1 Ser-473 phosphorylation under stress.

Fusobacterium nucleatum Infection Induces Malignant Proliferation of Esophageal Squamous Cell Carcinoma Cell by Putrescine Production.

Esophageal squamous cell carcinoma (ESCC) is a malignant upper digestive tract cancer, and its pathogenesis and etiology are poorly understood. Because gut microbes commonly impact progression, metastasis, and immunotherapy responses in colorectal cancer (CRC), the roles of the esophageal microbiota in ESCC have gradually drawn attention. As reported previously, Fusobacterium nucleatum (Fn), the notable "culprit" of CRC, can also influence the prognosis of ESCC in clinical studies. However, thus far, the underlying mechanism is unclear. In this study, 73 Chinese ESCC samples were collected. In those clinical samples, the abundance of Fn was found to be higher in tumors than in adjacent normal tissues, and a high abundance of Fn was correlated with shorter survival. Furthermore, using in vitro experiments, we demonstrated that Fn can invade ESCC cells, enhancing their proliferation capacity. The mechanism study revealed that Fn can produce high levels of putrescine after invasion, which disturbs polyamine metabolism and promotes the malignant proliferation of ESCC cells. In conclusion, Fn infection was found in Chinese ESCC tumor tissue samples and may promote ESCC progression by disturbing the polyamine metabolism pathway. IMPORTANCE Nowadays, the complex and varied interactions between microbes and human body are known to be crucial for maintaining the health of the human body. However, knowledge concerning the influence of esophageal microbes on the progression of esophageal squamous cell carcinoma is limited. Here, in our study, we confirmed that F. nucleatum can invade ESCC cells and consequently promote their proliferation, suggesting that esophageal microbes likely influence the progression of ESCC in clinical settings. Because the esophagus connects the oral cavity and stomach, acting as a canal for transporting foods, its special physical location makes it easily exposed to microorganisms. Thus, it is necessary to explore the interaction between esophageal microbes and ESCC.

Integrated multi-omics profiling yields a clinically relevant molecular classification for esophageal squamous cell carcinoma.

Integrated molecular analysis of human cancer has yielded molecular classification for precise management of cancer patients. Here, we analyzed the whole genomic, epigenomic, transcriptomic, and proteomic data of 155 esophageal squamous cell carcinomas (ESCCs). Multi-omics analysis led to the classification of ESCCs into four subtypes: cell cycle pathway activation, NRF2 oncogenic activation, immune suppression (IS), and immune modulation (IM). IS and IM cases were highly immune infiltrated but differed in the type and distribution of immune cells. IM cases showed better response to immune checkpoint blockade therapy than other subtypes in a clinical trial. We further developed a classifier with 28 features to identify the IM subtype, which predicted anti-PD-1 therapy response with 85.7% sensitivity and 90% specificity. These results emphasize the clinical value of unbiased molecular classification based on multi-omics data and have the potential to further improve the understanding and treatment of ESCC.

p14(ARF) inhibits the functions of adenovirus E1A oncoprotein.

The tumour suppressor ARF (alternative reading frame) is one of the most important oncogenic stress sensors. ARF provides an 'oncogenic checkpoint' function through both p53-dependent and p53-independent mechanisms. In the present study, we demonstrate a novel p53-independent interaction between p14(ARF) and the adenovirus oncoprotein E1A. p14(ARF) inhibits E1A transcriptional function and promotes ubiquitination-dependent degradation of E1A. p14(ARF) overexpression relocalizes E1A into the nucleolus and inhibits E1A-induced cellular DNA replication independent of p53. Knockdown of endogenous p14(ARF) increases E1A transactivation. In addition, E1A can competitively inhibit ARF-Mdm2 (murine double minute 2) complex formation. These results identify a novel binding partner of p14(ARF) and reveal a mutually inhibitory interaction between p14(ARF) and E1A. We speculate that the ARF-E1A interaction may represent an additional host defence mechanism to limit viral replication. Alternatively, the interaction may allow adenovirus to sense the functional state of p53 in host cells, and fine-tune its own replication activity to prevent the triggering of a detrimental host response.

Comprehensive characterization of posttranscriptional impairment-related 3'-UTR mutations in 2413 whole genomes of cancer patients.

The 3' untranslated region (3'-UTR) is the vital element regulating gene expression, but most studies have focused on variations in RNA-binding proteins (RBPs), miRNAs, alternative polyadenylation (APA) and RNA modifications. To explore the posttranscriptional function of 3'-UTR somatic mutations in tumorigenesis, we collected whole-genome data from 2413 patients across 18 cancer types. Our updated algorithm, PIVar, revealed 25,216 3'-UTR posttranscriptional impairment-related SNVs (3'-UTR piSNVs) spanning 2930 genes; 24 related RBPs were significantly enriched. The somatic 3'-UTR piSNV ratio was markedly increased across all 18 cancer types, which was associated with worse survival for four cancer types. Several cancer-related genes appeared to facilitate tumorigenesis at the protein and posttranscriptional regulation levels, whereas some 3'-UTR piSNV-affected genes functioned mainly via posttranscriptional mechanisms. Moreover, we assessed immune cell and checkpoint characteristics between the high/low 3'-UTR piSNV ratio groups and predicted 80 compounds associated with the 3'-UTR piSNV-affected gene expression signature. In summary, our study revealed the prevalence and clinical relevance of 3'-UTR piSNVs in cancers, and also demonstrates that in addition to affecting miRNAs, 3'-UTR piSNVs perturb RBPs binding, APA and m6A RNA modification, which emphasized the importance of considering 3'-UTR piSNVs in cancer biology.

Role of Epidermal Growth Factor Receptor-Specific CAR-T Cells in the Suppression of Esophageal Squamous Cell Carcinoma.

ESCC is a highly malignant tumor, and its morbidity and mortality in China account for more than 50% of the world's total rates. As effective treatments are lacking, the 5-year survival rate of patients does not exceed 30%. CAR-T-cell-based immunotherapy has emerged as one of the most promising cancer treatments; however, there are relatively fewer reports regarding its application for ESCC. In this study, we conducted large-sample whole-genome sequencing (WGS) and RNA-seq analysis of patients with ESCC from China to examine the feasibility of EGFR-targeting CAR-T cells in the treatment of ESCC. We found much higher levels of EGFR gene amplification and overexpression in tumors than in the normal tissues, indicating that EGFR could be a promising target of CAR-T-cell-based immunotherapy in ESCC. Therefore, we tested EGFR-targeting CAR-T cells for lytic activity against ESCC cells as a model to establish cellular immunotherapy for ESCC. Five types of CAR-T cells targeting EGFR were constructed, two of which, CAR1-T and CAR2-T, showed a strong cytotoxicity against ESCC in in vitro and in vivo experiments. The results of this study suggest that CAR1-T and CAR2-T have the potential to be used for anti-ESCC immunotherapy in clinics.

A Radiomics Nomogram for Non-Invasive Prediction of Progression-Free Survival in Esophageal Squamous Cell Carcinoma.

To construct a prognostic model for preoperative prediction on computed tomography (CT) images of esophageal squamous cell carcinoma (ESCC), we created radiomics signature with high throughput radiomics features extracted from CT images of 272 patients (204 in training and 68 in validation cohort). Multivariable logistic regression was applied to build the radiomics signature and the predictive nomogram model, which was composed of radiomics signature, traditional TNM stage, and clinical features. A total of 21 radiomics features were selected from 954 to build a radiomics signature which was significantly associated with progression-free survival (p < 0.001). The area under the curve of performance was 0.878 (95% CI: 0.831-0.924) for the training cohort and 0.857 (95% CI: 0.767-0.947) for the validation cohort. The radscore of signatures' combination showed significant discrimination for survival status. Radiomics nomogram combined radscore with TNM staging and showed considerable improvement over TNM staging alone in the training cohort (C-index, 0.770 vs. 0.603; p < 0.05), and it is the same with clinical data (C-index, 0.792 vs. 0.680; p < 0.05), which were confirmed in the validation cohort. Decision curve analysis showed that the model would receive a benefit when the threshold probability was between 0 and 0.9. Collectively, multiparametric CT-based radiomics nomograms provided improved prognostic ability in ESCC.